The long-term goal of Project 3 is to define mechanisms that cause endothelial NO synthase (eNOS) to dysfunction and generate (in addition to NO) reactive oxygen and nitrogen species (oxidants) that cause oxidative damage in inflammatory diseases. We hypothesize that two unique aspects of eNOS are critical for its proper function and also predispose eNOS to be susceptible to dysfunction leading to oxidative damage: 1) eNOS redox cycling of its cofactor tetrahydrobiopterin (H4B);and 2) eNOS protein-protein interactions. Mechanisms that control these aspects are unclear and are the focus of this proposal. We will determine mechanisms at a molecular, kinetic, and structural level and ultimately translate them to cellular and clinical studies. AIM 1 will test if inefficient H4B redox cycling occurs in eNOS and predisposes it to uncoupled NO synthesis and oxidant formation. We will: (i) Quantify H4B redox cycling in eNOS and develop a kinetic model to explain oxidant formation, (ii) Test eNOS mutants for improved H4B redox cycling and less oxidant formation, and (iii) Measure metabolic indices associated with enhanced eNOS uncoupling to determine if they are linked with increased systemic indices of nitrative stress and prevalence for cardiovascular disease in humans. AIM 2 will investigate how eNOS function is controlled by interactions with heat shock protein 90 (HSP90). We will: (i) Test the hypothesis that HSP90 improves eNOS function by affecting specific catalytic steps in eNOS. (ii) Utilize fluorescence labeling, mass spectrometry, calorimetry, and protein crystallography to investigate HSP90 and Cav1 binding to eNOS, conformational changes that accompany binding, and mechanism for their effects on eNOS. (iii) Design site-specific mutants of eNOS with modified responses to Cavl and HSP90 and test their function, (iv) Determine mechanisms that enable phosphorylation and activation of eNOS when bound in a trimeric complex with HSP90 and the kinase Akt1. and (v) Investigate if a single nucleotide polymorphism in eNOS (Asp298Glu) associated with increased cardiovascular risk impacts HSP90 interactions with eNOS. Collectively, the proposed studies will provide a deep mechanistic understanding of factors regulating eNOS fucntion and dysfunction in cardiovascular disease.